Basic fibroblast growth factor (FGF2 or bFGF) is a growth factor secreted from various cells, which is deeply involved in the cell proliferation and differentiation in developmental stages and shows high expression during tissue repair and in cancer tissues in adults.
While human FGF2 has plural isoforms, only an isoform having the lowest molecular weight is extracellularly secreted. This isoform is an about 18 kDa protein consisting of 154 amino acids, which is free of a sugar chain and has a basic isoelectric point of 9.4. While the function of high molecular weight isoforms (22, 22.5, 24, 34 kD) of FGF2 with different open reading frames is not clear as yet, they are considered to have a nuclear localization signal and function in the nucleus.
The human FGF family protein is known to include 22 kinds from FGF1 to FGF23 (FGF15 and FGF19 are now unified as FGF19 since they are the same molecules). By phylogenetic analysis, FGF2 is classified into FGF1 subfamily together with FGF1. The homology of amino acid sequence with FGF1 is the highest of all FGFs, and its value is 55%. FGF receptor (FGFR) is a receptor tyrosine kinase and classified into 4 subtypes. Each of FGFR1-3 is known to include b and c isoforms. FGF2 binds to FGFR1b, FGFR1c, FGFR2c and FGFR3c, and FGFR4 therefrom to dimerize these receptors.
Mouse fibroblast (NIH-3T3 cell) expresses FGFR1 on the cellular membrane surface. FGFR1 is known to be activated when bound to human FGF2. When FGF2 is bound to FGFR1, MAP kinase (mitogen-activated protein kinase) pathway, PIK3 (phosphatidylinositol 3-kinase)/AKT1 (protein kinase B) pathway and the like are activated via FRS2 (Fibroblast growth factor receptor substrate 2), Grb2 (growth factor receptor-bound protein 2), SOS, and finally, expression of various cytokine and receptor genes such as VEGF (vascular endothelial growth factor precursor)-A, VEGF-C, HGF (hepatocyte growth factor), angiopoietin-2, VEGFR, PDGFR-α (platelet-derived growth factor beta receptor-α) and the like is induced.
FGF2 has a heparin binding region and, like other FGFs, is bound to heparin and heparan sulfate. It is generally considered that FGF2 secreted from a cell is bound to a heparan sulfate of an extracellular matrix, concentrated, and protected from protease. To function as a ligand, FGF2 needs to be liberated from the extracellular matrix bound thereto, in which FGF-BP (FGF-binding protein) is reported to be involved to aid induction to FGFR.
FGF2 is known to have a strong growth, cell migration-promoting effect for vascular endothelial cells, and be deeply involved in the angiogenesis of tumor tissues. A particularly high FGF2 serum concentration in tumor with many blood vessels, for example, renal cancer and the like, has been reported, and FGF2 is present in various other tumors such as prostate cancer, breast cancer, lung cancer and the like.
Factors such as FGF1, VEGF, TNF-α (tumor necrosis factor-α), PDGF, EGF (epidermal growth factor), MMP (matrix metallopeptidase), angiogenin and the like are involved in angiogenesis besides FGF2. These factors are secreted from tumor, angioblastic cells, supporting cells and the like, and contribute to angiogenesis as growth factors of autocrine or paracrine. However, FGF2 is different from other factors since it acts not only on vascular endothelial cells but also mesenchymal cells surrounding the endothelial cells, such as smooth muscle cell and the like. In other words, it is considered that FGF2 stimulates mesenchymal cell to promote expression of PDGF, PDGFR, VEGF, HGF and the like, and these factors enhance direct growth of vascular endothelial cells.
At present, many attempts have been made to develop a drug that inhibits abnormal angiogenesis in a tumor tissue to block a nutrient supply pathway to a tumor tissue. There is a drug actually used in clinical situations such as a humanized anti-VEGF monoclonal antibody (avastin (registered trade mark)) developed by Genentech, which has been confirmed to show an effect for colon cancer and non-small cell lung cancer. However, a strong antitumor drug has not been developed yet. Many of these drugs target VEGF and PDGF, and are expected to block the initial stages of abnormal angiogenesis by targeting FGF2 that functions at more upstream.
Abnormal angiogenesis is also involved in, besides tumor, diseases such as chronic inflammations (e.g., periodontal disease, scleroderma, neovascular glaucoma, arthritis and the like), psoriasis, age-related macular degeneration and the like.
On the other hand, an attempt has been made to use the strong angiogenic action of FGF2 for the treatment of occlusive vascular disorders and wound healing. In fact, the human FGF2 preparation (FIBLAST (registered trade mark) Spray) of Kaken Pharmaceutical Co., Ltd. has already been approved and sold as a drug for promoting wound healing.
While FGF2 is known to have an osteogenesis promoting effect, it is also attracting attention as a bone resorption-promoting factor since it is involved in articular destruction in chronic rheumatoid arthritis patients. In chronic rheumatoid arthritis characterized by autoimmune arthritis, the number of osteoclasts increases to promote bone resorption, which in turn progresses bone destruction.
FGF2 stimulates mesenchymal cell to promote expression of inflammatory cytokines and growth factors such as PDGF, PDGFR, VEGF, HGF and the like, as well as promotes angiogenesis and induces bone destruction. It has been clarified that FGF2 is a key molecule involved in significant pathology in chronic rheumatoid arthritis (non-patent document 1).
Osteoprotegerin (OPG) is a decoy receptor of an osteoclast inducer, RANKL, and is known to antagonize RANK and suppress differentiation induction into osteoclast and the function thereof (non-patent document 2). OPG produced from synovial cell is also known to be suppressed by stimulation of FGF2 (non-patent document 3). Furthermore, FGF2 encourages coupling of osteoblasts and osteoclast precursor cells by inducing high expression of RANKL by osteoblasts, as a result of which promotes differentiation into osteoclast and activation (non-patent document 4).
Once the function of FGF2 can be controlled, the effect as a therapeutic drug for articular destruction via activation of osteoclast should be sufficiently expected. In fact, direct articular administration of an anti-FGF2 neutralizing antibody to AIA model rats is known to mitigate the symptom. However, a suppressive effect on the onset thereof is small, and particularly, a healing effect by the administration after the onset has not been confirmed (non-patent document 5).
In recent years, applications of RNA aptamers to medicaments, diagnostic reagents, and test reagents have been drawing attention; some RNA aptamers have already been in clinical study stage or in practical use. In December 2004, the world's first RNA aptamer drug, Macugen, was approved as a therapeutic drug for age-related macular degeneration in the US. An RNA aptamer refers to an RNA that binds specifically to a target substance such as a protein, and can be prepared using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (see Patent documents 1-3). In the SELEX method, an RNA that binds specifically to a target substance is selected from an RNA pool with about 1014 different nucleotide sequences. The RNA structure used has a random sequence of about 40 residues, which is flanked by primer sequences. This RNA pool is allowed to be assembled with a target substance, and only the RNA that has bound to the target substance is collected using a filter and the like. The RNA collected is amplified by RT-PCR, and this is used as a template for the next round. By repeating this operation about 10 times, an RNA aptamer that binds specifically to the target substance can sometimes be acquired.
Patent document 4 describes an aptamer that binds to FGF2, which is obtained by the above-mentioned SELEX method. However, the sequences of the aptamers are different from those of the aptamers specifically shown in the present specification. In addition, this document does not suggest the aptamers specifically shown in the present specification.